Tumor Protein Synthesis Research Articles

Auranofin and reactive oxygen species inhibit protein synthesis and regulate the level of the PLK1 protein in Ewing sarcoma cells.

Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.

Preliminary exploration of amide proton transfer weighted imaging in differentiation between benign and malignant bone tumors.

To explore the value of 3D amide proton transfer weighted imaging (APTWI) in the differential diagnosis between benign and malignant bone tumors, and to compare the diagnostic performance of APTWI with traditional diffusion-weighted imaging (DWI). Patients with bone tumors located in the pelvis or lower limbs confirmed by puncture or surgical pathology were collected from January 2021 to July 2023 in the First Affiliated Hospital of Zhengzhou University. All patients underwent APTWI and DWI examinations. The magnetization transfer ratio with asymmetric analysis at the frequency offset of 3.5 ppm [MTRasym(3.5 ppm)] derived by APTWI and the apparent diffusion coefficient (ADC) derived by DWI for the tumors were measured. The Kolmogorou-Smirnou and Levene normality test was used to confirm the normal distribution of imaging parameters; and the independent sample t test was used to compare the differences in MTRasym(3.5 ppm) and ADC between benign and malignant bone tumors. In addition, the receiver operating characteristic (ROC) curve was used to evaluate the diagnostic performance of different imaging parameters in differentiation between benign and malignant bone tumors. P<0.05 means statistically significant. Among 85 bone tumor patients, 33 were benign and 52 were malignant. The MTRasym(3.5 ppm) values of malignant bone tumors were significantly higher than those of benign tumors, while the ADC values were significantly lower in benign tumors. ROC analysis shows that MTRasym(3.5 ppm) and ADC values perform well in the differential diagnosis of benign and malignant bone tumors, with the area under the ROC curve (AUC) of 0.798 and 0.780, respectively. Combination of MTRasym(3.5 ppm) and ADC values can further improve the diagnostic performance with the AUC of 0.849 (sensitivity = 84.9% and specificity = 73.1%). MTRasym(3.5 ppm) of malignant bone tumors was significantly higher than that of benign bone tumors, reflecting the abnormal increase of protein synthesis in malignant tumors. APTWI combined with DWI can achieve a high diagnostic efficacy in differentiation between benign and malignant bone tumors.

Tumour-specific and organ-specific protein synthesis rates in patients with pancreatic cancer.

BackgroundLiving tissues maintain a fine balance between protein synthesis and protein breakdown rates. Animal studies indicate that protein synthesis rates are higher in organs when compared with skeletal muscle tissue. As such, organ and tumour protein synthesis could have major effects on whole‐body protein metabolism in wasting disorders such as cancer cachexia. We aimed to assess protein synthesis rates in pancreatic tumour tissue and healthy pancreas, liver, and skeletal muscle tissue in vivo in humans.MethodsIn eight patients with pancreatic cancer undergoing pancreaticoduodenectomy, primed continuous infusions with L‐[ring‐13C6]phenylalanine and L‐[3,5‐2H2]tyrosine were started prior to surgery and continued throughout the surgical procedures. During surgery, plasma samples and biopsies from the pancreas, pancreatic tumour, liver, and vastus lateralis muscle were taken. Post‐absorptive fractional protein synthesis rates were determined by measuring incorporation of labelled L‐[ring‐13C6]phenylalanine in tissue protein using the weighed plasma L‐[ring‐13C6]phenylalanine enrichments as the precursor pool.ResultsFive male patients and three female patients with a mean age of 67 ± 2 years were included into this study. Plasma L‐[ring‐13C6]phenylalanine enrichments (6–9 mole per cent excess) did not change during surgery (P = 0.60). Pancreatic tumour protein synthesis rates were 2.6‐fold lower than surrounding pancreatic tissue protein synthesis rates (0.268 ± 0.053 vs. 0.694 ± 0.228%/h, respectively; P = 0.028) and 1.7‐fold lower than liver protein synthesis rates (0.268 ± 0.053 vs. 0.448 ± 0.043%/h, respectively; P = 0.046). Among healthy organ samples, protein synthesis rates were 20‐fold and 13‐fold higher in pancreas and liver, respectively, compared with skeletal muscle tissue (0.694 ± 0.228 and 0.448 ± 0.043 vs. 0.035 ± 0.005%/h, respectively; P < 0.05).ConclusionsLiver and pancreas tissue protein synthesis rates are higher when compared with pancreatic tumour and skeletal muscle tissue protein synthesis rates and can, therefore, strongly impact whole‐body protein metabolism in vivo in humans.

Leucine-rich diet alters the 1H-NMR based metabolomic profile without changing the Walker-256 tumour mass in rats.

BackgroundCachexia is one of the most important causes of cancer-related death. Supplementation with branched-chain amino acids, particularly leucine, has been used to minimise loss of muscle tissue, although few studies have examined the effect of this type of nutritional supplementation on the metabolism of the tumour-bearing host. Therefore, the present study evaluated whether a leucine-rich diet affects metabolomic derangements in serum and tumour tissues in tumour-bearing Walker-256 rats (providing an experimental model of cachexia).MethodsAfter 21 days feeding Wistar female rats a leucine-rich diet, distributed in L-leucine and LW-leucine Walker-256 tumour-bearing groups, we examined the metabolomic profile of serum and tumour tissue samples and compared them with samples from tumour-bearing rats fed a normal protein diet (C – control; W – tumour-bearing groups). We utilised 1H-NMR as a means to study the serum and tumour metabolomic profile, tumour proliferation and tumour protein synthesis pathway.ResultsAmong the 58 serum metabolites examined, we found that 12 were altered in the tumour-bearing group, reflecting an increase in activity of some metabolic pathways related to energy production, which diverted many nutrients toward tumour growth. Despite displaying increased tumour cell activity (i.e., higher Ki-67 and mTOR expression), there were no differences in tumour mass associated with changes in 23 metabolites (resulting from valine, leucine and isoleucine synthesis and degradation, and from the synthesis and degradation of ketone bodies) in the leucine-tumour group. This result suggests that the majority of nutrients were used for host maintenance.ConclusionA leucine rich-diet, largely used to prevent skeletal muscle loss, did not affect Walker 256 tumour growth and led to metabolomic alterations that may partially explain the positive effects of leucine for the whole tumour-bearing host.

Assessment of time from onset of first symptoms to the final diagnosis of multiple myeloma (MM) - possible risks and future solutions

Assessment of time from onset of first symptoms to the final diagnosis of multiple myeloma (MM) - possible risks and future solutions

KinaseAKTcontrols innate immune cell development and function

The critical roles of kinase AKT in tumour cell proliferation, apoptosis and protein synthesis have been widely recognized. But AKT also plays an important role in immune modulation. Recent studies have confirmed that kinase AKT can regulate the development and functions of innate immune cells (neutrophil, macrophage and dendritic cell). Studies have shown that different isoforms of kinase AKT have different effects in regulating immunity-related diseases, mainly through the mammalian target of rapamycin-dependent or -independent pathways. The purpose of this review is to illustrate the immune modulating effects of kinase AKT on innate immune cell development, survival and function.

In Vivo PET/CT in a Human Glioblastoma Chicken Chorioallantoic Membrane Model: A New Tool for Oncology and Radiotracer Development

For many years the laboratory mouse has been used as the standard model for in vivo oncology research, particularly in the development of novel PET tracers, but the growth of tumors on chicken chorioallantoic membrane (CAM) provides a more rapid, low cost, and ethically sustainable alternative. For the first time, to our knowledge, we demonstrate the feasibility of in vivo PET and CT imaging in a U87 glioblastoma tumor model on chicken CAM, with the aim of applying this model for screening of novel PET tracers. U87 glioblastoma cells were implanted on the CAM at day 11 after fertilization and imaged at day 18. A small-animal imaging cell was used to maintain incubation and allow anesthesia using isoflurane. Radiotracers were injected directly into the exposed CAM vasculature. Sodium (18)F-fluoride was used to validate the imaging protocol, demonstrating that image-degrading motion can be removed with anesthesia. Tumor glucose metabolism was imaged using (18)F-FDG, and tumor protein synthesis was imaged using 2-(18)F-fluoro-l-tyrosine. Anatomic images were obtained by contrast-enhanced CT, facilitating clear delineation of the tumor, delineation of tracer uptake in tumor versus embryo, and accurate volume measurements. PET imaging of tumor glucose metabolism and protein synthesis was successfully demonstrated in the CAM U87 glioblastoma model. Catheterization of CAM blood vessels facilitated dynamic imaging of glucose metabolism with (18)F-FDG and demonstrated the ability to study PET tracer uptake over time in individual tumors, and CT imaging improved the accuracy of tumor volume measurements. We describe the novel application of PET/CT in the CAM tumor model, with optimization of typical imaging protocols. PET imaging in this valuable tumor model could prove particularly useful for rapid, high-throughput screening of novel radiotracers.

SBDS-deficiency results in specific hypersensitivity to Fas stimulation and accumulation of Fas at the plasma membrane

Shwachman-Diamond syndrome (SDS) is an inherited disorder characterized by reduced cellularity in the bone marrow and exocrine pancreas. Most patients have mutations in the SBDS gene, whose functions are unknown. We previously showed that cells deficient in the SBDS protein are characterized by accelerated apoptosis and Fas hypersensitivity, suggesting that the protein might play an important role in Fas-mediated apoptosis. To study the mechanism of Fas hypersensitivity, we compared shRNA-mediated SBDS-knockdown HeLa cells and SDS marrow CD34+ cells for their sensitivity to several groups of apoptosis inducers. Marked hypersensitivity was noticed in response to Fas stimulation, but not to tumor necrosis factor-alpha, DNA-damaging agents, transcription inhibition or protein synthesis inhibition. To identify the Fas signaling factors that cause hypersensitivity, we analyzed the expression of the pathway's proteins. We found that Fas accumulated at the plasma membrane in SBDS-knockdown cells with corresponding expression of Fas transcript 1, the main Fas transcript which contains both the transmembrane domain and the death domain. However, the total levels of Fas protein and mRNA were comparable to controls, and Fas internalization occurred normally. Expression of FADD, caspase-8 and -3 were not elevated and the pathway inhibitors: ERK, c-FLIP and XIAP were not decreased. These results suggest that SBDS loss results in abnormal accumulation of Fas at the plasma membrane, where it sensitizes the cells to stimulation by Fas ligand.

Enhanced antiglioma activity of chimeric HCMV/HSV-1 oncolytic viruses

Oncolytic herpes simplex virus (HSV)-1 gamma(1)34.5-deletion mutants (Deltagamma(1)34.5 HSV) are promising agents for tumor therapy. The attenuating mutation renders the virus aneurovirulent but also limits late viral protein synthesis and efficient replication in many tumors. We tested whether one function of gamma(1)34.5 gene, which mediates late viral protein synthesis through host protein kinase R (PKR) antiviral response evasion, could be restored, without restoring the neurovirulence. We have previously reported the construction of two chimeric Deltagamma(1)34.5 HSV vectors (chimeric HSV), C130 and C134, which express the human cytomegalovirus (HCMV) PKR-evasion genes TRS1 and IRS1, respectively. We now demonstrate the following. The HCMV/HSV-1 chimeric viruses (i) maintain late viral protein synthesis in the human malignant glioma cells tested (D54-MG, U87-MG and U251-MG); (ii) replicate to higher titers than Deltagamma(1)34.5 HSV in malignant glioma cells in vitro and in vivo; (iii) are aneurovirulent; and (iv) are superior to other Deltagamma(1)34.5 HSV with both improved reduction of tumor volumes in vivo, and improved survival in two experimental murine brain tumor models. These findings demonstrate that transfer of HCMV IRS1 or TRS1 gene into Deltagamma(1)34.5 HSV significantly improves replication in malignant gliomas without restoring wild-type neurovirulence, resulting in enhanced tumor reduction and prolonged survival.

Stimulation of protein synthesis in human tumours by parenteral nutrition: Evidence for modulation of tumour growth

Eighteen patients with localized colorectal carcinoma were randomized to receive intravenous nutrition or to be fasted during the 24 h before surgery. Protein synthesis, an index of tumour growth, was then measured by the incorporation of [13C]leucine into tumour protein immediately before surgery. The mean (s.e.m.) rate of tumour protein synthesis in patients receiving nutrition (42.7(3.5) per cent per day) was 89 per cent higher than the rate in the fasted group (22.6(1.9) per cent per day) (P = 0.002). As tumours consist of a variety of different cell types, in vitro rates of protein synthesis were measured in malignant cells isolated from colorectal tumours and cultured with autologous serum obtained from the patient in either the fasted or the fed state. There was a mean increase of 81 per cent in protein synthesis when fed rather than fasted serum was used (P less than 0.02), indicating that the malignant cells themselves respond to nutrient supply. This increase in tumour protein synthesis provides the first evidence in vivo that the exogenous supply of nutrients can modulate the rate of growth of a human tumour.

Serum Free Light Chain (SFLC) Concentration Kinetics in Patients Receiving Bortezomib: Temporary Inhibition of Protein Synthesis and Early Biomarker for Disease Response.

Monoclonal intact immunoglobulin is detectable in the serum of approximately 80% of myeloma patients while SFLC are found to be abnormal in the serum of greater than 90% of patients. One advantage of monitoring SFLC is that they have a half-life of clearance from the serum of 2–6 hours, compared with 20 days for IgG, and can therefore reveal the response to treatment more rapidly. The aim of this study is to determine whether SFLC measurements allow earlier assessment of response to treatment with bortezomib with possible implications to the design and cost of treatment strategy.Patients & Methods Serial serum samples were collected for simultaneous measurement of SFLC and serum paraprotein. Data from 8 patients receiving 2–6 cycles of bortezomib, on compassionate basis, to treat multiply treated myeloma at various stages of relapse are presented. Bortezomib was administered at a dose of 1.3mg/m2 on days 1,4,8 and11. Samples were kept frozen until thawed for SFLC measurement (using Freelite kits; Binding Site, UK on an Olympus Chemistry analyser).Results & Discussion Six of 8 patients showed a response to treatment by EBMT/Blade criteria (>25% fall in paraprotein). In 3 of these patients the tumour SFLC showed a fall and rapid recovery within 10–20 days of a treatment cycle. These patients were monitored over multiple treatment cycles and showed repeated falls in SFLC co-incident with treatment and recovery in between. This appears to correspond with the mechanism of action and biological half life of proteosome inhibition. Recovery of SFLC when seen is rapid (doubling in <10 days). This pattern of tumour response would only be observable with frequent sampling and a tumour marker rapidly cleared from the serum and we believe this is the first reported observation in myeloma patients. In comparison to SFLC, the intact immunoglobulin monoclonal protein did not show the same peaks and troughs. It is likely that the pattern of initial SFLC response is an early indication of tumour response or resistance. Generally the SFLC levels indicated disease response earlier than the immunoglobulin assays. In two of these patients, the response was seen significantly earlier (30 and 70 days). In one remaining patient, serum paraprotein showed disease response whereas SFLC showed no response and subsequently disease progression.Conclusion: We conclude that monitoring SFLC provides a unique opportunity to follow the kinetics of tumour kill, which is obscured by the slow clearance of intact immunoglobulin monoclonal proteins. The pattern of fluctuation in the SFLC levels could suggest a temporary inhibition of tumour protein synthesis rather than tumour kill and re-growth. While extension of this study is ongoing, it does indicate that SFLC can be used as a biomarker to assess response to treatment significantly earlier allowing relevant changes of treatment strategy. This also may have major bearing on cost of treatment and utilisation of resources.

Effect of glucagon on protein synthesis in human rectal cancer in situ.

The purpose of this study was to determine the effect of glucagon or placebo on the rate of tumor fractional protein synthesis in situ in patients with localized rectal cancer who were not malnourished, demonstrated normal glucagon concentrations, and could therefore be used as a model to study the glucagon effect. Cancer cachexia is associated with an increased concentration of counterregulatory hormones, including glucagon. This altered hormonal milieu may not only contribute to malnutrition, but also promote tumor growth, because previous experimental work suggests that glucagon can cause human colorectal tumor cells to proliferate. Corresponding mechanisms in vivo have, thus far, not been investigated. Advanced mass spectrometry techniques (capillary gas chromatography [GC]/combustion isotope ratio mass spectrometry [IRMS]) were used to determine directly the incorporation rate of 1-[13C]-leucine into tissue protein. Because GC/IRMS requires only a small sample volume, three consecutive endoscopic biopsies could be obtained from the same tumor to determine isotopic enrichments at baseline, after a 4-hour glucagon infusion (3 ng/kg/min), or after placebo. In patients with localized rectal cancer, glucagon caused the tumor fractional protein synthetic rate to double (2.25+/-0.49 %/hr, p < 0.05 vs. 1.16+/-0.30 basal). In contrast, tumor protein synthesis declined over time in controls (placebo) (0.67+/-0.09 %/hr, p < 0.05 vs. 1.11+/-0.16 basal). Tumor protein synthesis and growth can be stimulated by glucagon in situ. Therefore, elevated glucagon concentrations in cachectic cancer patients should be considered detrimental and attempts made to prevent this specific response of the body to the malignant disease.

11C-tyrosine position emission tomography and 1H magnetic resonance spectroscopy of the response of brain gliomas to radiotherapy.

We monitored 10 patients with unresected (9) or partially resected (1) supratentorial gliomas with 11C-tyrosine position emission tomography (TYR-PET) before and after radiotherapy. TYR-PET tumour volumes were measured using a threshold technique. In seven patients the tumour volume decreased after radiotherapy, although all gliomas persisted on TYR-PET images. In eight patients the tumour protein synthesis rate (PSR) was calculated using a dynamic study protocol in combination with a PATLAK analysis. There were no changes in PSR after radiotherapy, but the PSR was calculated on the remaining tumour volume using the same threshold technique as before therapy, i.e. the decrease in tumour volume was not taken into account. In eight patients the PET data were compared with magnetic resonance spectroscopic imaging (1H-MRSI) performed simultaneously. Although there was no statistically significant correlation between TYR-PET volume changes and 1H-MRSI choline level we observed a simultaneous decrease in volume and choline in four patients.

Effect of methionine-free total parenteral nutrition and insulin-like growth factor I on tumor growth in rats.

The objectives of this study were to evaluate the effect of insulin-like growth factor I (IGF-I) on the fractional synthesis rate (FSR) of muscle and whole body protein breakdown rate (WPBR) during methionine-free total parenteral nutrition (MTPN). We also determined whether the inhibition of endogenous methionine availability reduced tumor protein synthesis. AH109A hepatoma cells were inoculated onto the backs of Donryu rats on day 0. On day 10, the rats were catheterized for TPN and assigned to one of four groups: 1) standard TPN (STPN), 2) STPN + IGF-I, 3) MTPN, or 4) MTPN + IGF-I. The addition of IGF-I to MTPN reduced the loss of body weight by both increasing muscle FSR and reducing WPBR. The tumor FSR did not differ between MTPN + IGF-I and MTPN. The methionine extraction ratio from the liver was negative with MTPN + IGF-I but positive in the other groups. We concluded that IGF-I blockage of endogenous methionine release from peripheral protein sites was associated with a shift to liver-derived methionine, with no change in tumor growth in MTPN-treated rats.

Determination of protein synthesis in human rectal cancer in situ by continuous [1-13C]leucine infusion.

Previous studies on human colorectal tumor protein synthesis in situ relied on techniques that required intra- or perioperative sampling to obtain a sufficient biopsy size. The purpose of the present study was to develop a new technique by use of new mass spectrometry equipment [capillary gas chromatography (GC)-combustion isotope ratio mass spectrometry (IRMS)], which allows reduction of the necessary sampling size. Thereby, tumor sampling could be done via conventional rectosigmoidoscopy, excluding the need for further disturbing invasive measures. Fifteen postabsorptive patients with localized rectal cancer received a primed-constant infusion of [1-13C]leucine (0.16 mumol.kg-1.min-1 constant, 9.6 mumol/kg prime). Forceps biopsies were taken after 3 and 6 h. In five subjects, tumor tissue and normal mucosa were studied simultaneously. Determination of protein-bound leucine enrichment was done by GC-IRMS, and GC-quadrupole MS was used to determine tracer-to-tracee ratios (tracer/tracee) for free intracellular leucine. GC-MS data demonstrated achievement of a steady state in the precursor pool enrichment after 3 h of isotope infusion (tracer/tracee at 3 h: 6.34 +/- 0.46%, at 6 h: 6.58 +/- 0.38%). Calculation of tumor protein synthesis yielded a fractional synthetic rate (FSR) of 1.06 +/- 0.11%/h or 25.5 +/- 2.6%/day (range 12.0-37.1%/day). At any time, protein-bound leucine enrichment was significantly higher in tumor tissue than in normal mucosa of the same subject. However, protein synthetic rates were comparable (tumor: 1.09 +/- 0.20%/h, mucosa: 1.29 +/- 0.28%/h). Thus combined GC-combustion IRMS and GC-/quadrupole MS provide a simple, reliable, and minimally invasive method to determine tumor FSR in situ, thereby excluding interferences common to previous methods. Tumor and mucosa tissues are similar with respect to protein synthesis, but they apparently differ with respect to leucine extraction from the arterial blood.

Increased Whole-Body Protein and Glutamine Turnover in Advanced Cancer Is Not Matched by an Increased Muscle Protein and Glutamine Turnover

In the progress of cancer major disturbances in protein and glutamine metabolism have been observed. Muscle is the major protein pool and glutamine source in the body. The aim of this study was to investigate whether changes in whole-body protein and glutamine turnover, induced by cancer, are matched by similar changes in regional muscle metabolism. A MCA sarcoma was implanted subcutaneously in female Lewis rats. Rats were studied bearing small (5–15% of body weight) or large (15–30% of body weight) tumor loads and compared with sham-implanted free-fed and pair-fed controls. Body composition was determined by the distribution of an ip bolus of3H2O. With the rat under anesthesia a primed constant infusion ofl-[2,6-3H]phenylalanine andl-[3,4-3H]glutamine was given, and at steady state, whole-body, hindquarter-muscle, and tumor protein and glutamine turnover were calculated using compartment modeling. Anorexia was not observed in tumor-bearing rats. A small decrease in host carcass weight was observed in large-tumor-bearing rats by decreased fat mass. Whole-body protein turnover increased from 115 ± 14 (nmole × 100 g body weight−1× min−1) in free-fed control rats to 239 ± 29 in the large-tumor-bearing rats. Net tumor protein synthesis accounted for 28 ± 1 and 49 ± 1 nmole × 100 g body weight−1× min−1. Muscle protein breakdown increased in the small-tumor-bearing group and decreased to control values in the large-tumor-bearing rats. Whole-body glutamine turnover remained unchanged in the small-tumor-bearing animals (2481 ± 248 and 1996 ± 268 nmole × 100 g body weight−1× min−1in control and small-tumor-bearing rats, respectively) and increased by 25% in the large-tumor-bearing animals. In contrast, muscle glutamine turnover more than doubled in the small-tumor-bearing group but returned to control values in the large-tumor-bearing animals. The current study show that in the presence of a small tumor whole-body protein turnover increased and that this was in part related to protein turnover of the tumor. Muscle protein breakdown increased in these rats with a concomitant increase in glutamine production from the hindquarter. In animals bearing larger tumors whole-body glutamine turnover increased. This increase, however, was only for a small part caused by tumor metabolism. Muscle glutamine turnover even decreased. Therefore, the increase in whole-body glutamine turnover appears to be caused by increased turnover in visceral organs.

The sequential changes in DNA synthesis, glucose utilization, protein synthesis, and peripheral benzodiazepine receptor density in C6 brain tumors after chemotherapy to predict the response of tumors to chemotherapy

Monitoring therapy in patients with brain tumors is very difficult and unreliable. It has been shown that there is no good correlation between tumor sensitivity measured in vitro and in situ tumor response to therapies. Sequential changes in tumor size, number of DNA synthesizing cells (labelling index [LI]), glucose utilization (LCGU), protein synthesis (LCPS), and peripheral benzodiazepine receptor (PBR) density were examined after chemotherapy for seven days. This was done using antibromodeoxyuridine immunohistochemical stain and multiple tracer quantitative autoradiography in a C6 rat brain with an implanted glioma. On Day 10 after inoculation, the rats were divided into 5 experimental groups: (1) a nontreatment group (control Group 1); (2) a group received 5% dextrose intraarterial (IA) administration (control Group 2); (3) a group received 1,3-bis-(2-chloroethyl) nitrosourea (BCNU) intravenous (i.v.) administration (Group 3) (5% dextrose was solvent); (4) a group received BCNU IA administration (Group 4) (5% dextrose was solvent); and (5) a group received sarcosinamide chloroethyl nitrosourea (SarCNU) IA administration (Group 5) (solvent as for the BCNU group). Three treatments showed a significant decrease (P < 0.003) in tumor growth. The most effective treatment was BCNU IA and SarCNU IA was moderately effective. BCNU i.v. showed no effect on tumor growth when compared with the two control groups. The change in the peak LI correlated well with the peak LCGU. These parameters decreased markedly and significantly in both Group 4 and Group 5 from Day 1 after treatment. The rates of the decrease in these biologic factors also correlated well with a decrease in the tumor growth. The LCPS did not correlate with a decrease in the LI or LCGU. The dissociation constant (Kd) and densities of the receptors PBR (B max) did not change significantly in any of the treatment groups during the observation period. From the results presented, we concluded that changes in the LI and LCGU represent the most reliable parameters with which to predict the response or sensitivity of this glial tumor to the treatments applied. These data suggest that if changes in peak LCGU were measured in tumors using positron emission tomography, they might be instrumental in providing in vivo information about the sensitivity of a tumor to a given treatment without the need for repeated tumor biopsy.

Effect of glutamine supplementation on protein metabolism and glutathione in tumor-bearing rats.

Since tumor-bearing rats are deficient in glutamine, we investigated whether (1) glutamine and glutathione deficiency occur in tumor-bearing rats, (2) glutamine supplementation caused an increase of glutathione levels in host tissues and tumor, (3) glutamine enhances protein synthesis in host tissues, and (4) glutamine stimulated the tumor to synthesize protein and DNA. Male Donryu rats were randomized into four groups: (1) non-tumor-bearing rat (NTB) + standard total parenteral nutrition (STPN); (2) NTB + glutamine-supplemented TPN (GTPN); (3) tumor-bearing rat (TB) + STPN; (4) TB + GTPN. On day 0 AH109A rat hepatoma cells were subcutaneously injected into the backs of rats to induce tumor. The animals were maintained on TPN for 6 days from day 10 through day 15. On day 15, 1-14C-leucine was given by a 5-hour continuous infusion (2.0 microCi/h per rat) to determine the fractional synthesis rate and endogenous leucine production. The levels of glutamine and glutathione were measured by HPLC. the tumor DNA synthesis was estimated by bromodeoxyuridine labeling index. Tumor development led to a significant weight loss, but this weight loss was significantly lessened by glutamine supplementation because of an increase in muscle protein synthesis. Glutamine did not enhance tumor weight, protein, and DNA synthesis in the tumor. Tumor development caused a significant reduction of glutathione in the muscle, jejunum, and liver, but supplemented glutamine increased the levels of glutathione in the jejunum. Glutamine supplementation is beneficial in preventing deficiencies of glutamine and glutathione and in improving protein metabolism in tumor-bearing rats.

Effect of methionine-deprived total parenteral nutrition on tumor protein turnover in rats.

Previous studies have shown that a methionine-lacking diet inhibited tumor growth in rats. The aim of this study was to determine how methionine free total parenteral nutrition (MTPN) can result in the inhibition of tumor growth on tumor protein metabolism in rats. On day 0, AH109A rat ascites hepatoma cells were implanted subcutaneously into male Donryu rats (n = 68, body weight, 200-225 gm). On day 10, a catheter for total parenteral nutrition (TPN) was placed and either MTPN or standard TPN solution was given for 5 days. On day 15, 1-14C-leucine was infused continuously to measure tumor protein synthesis. Tumor proteolysis was calculated from tumor regional blood flow, using the 85Sr-microsphere injection method. 1) Tumor weight was reduced with MTPN. 2) MTPN did not affect tumor protein synthesis, probably because endogenous methionine production was increased with MTPN (87.3 +/- 13.5 mumole methionine/kg/hour vs. 218.6 +/- 29.5, P < 0.01); however, MTPN caused an increase of tumor proteolysis (2.68 +/- 0.53 mumole leucine/g/hour vs. 3.79 +/- 0.73, P < 0.05). The enhanced tumor protein breakdown contributed to the inhibition of tumor growth that was found with the rats given the methionine free diet.

Chemical induction of interleukin-8, a proinflammatory chemokine, in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 mumol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001-100 ng/ml), cycloheximide (CHX) (0.01-50 micrograms), lipopolysaccharide (0.1-100 micrograms/ml), tumor necrosis factor-alpha (TNF alpha) (3.13-50 ng/ml), or interleukin-1 alpha (IL-1 alpha) (1-182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNF alpha induced IL-8 production that coincided with significant cell cycle inhibition. IL-1 alpha had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31-10 micrograms/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNF alpha reduced the mitotic index by approximately 45%, slowed cell cycle progression by approximately 3.5 h, and induced a flat, albeit large, IL-8 response at concentrations > or = 12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.